Viable cell compositions, and methods related to same

ABSTRACT

Disclosed are viable cell compositions and related methods of preparation, maintenance and use. The viable cell composition can contain specified levels of cells, hydroxyethyl starch, and dimethylsulfoxide, and can be cryopreserved. The cryopreserved form of the composition can be thawed and combined with an aqueous liquid diluting medium to prepare a diluted viable cell composition that can contain specified, reduced levels of the dimethylsulfoxide and hydroxyethyl starch. The diluting medium can contain trehalose. The prepared, diluted viable cell composition can be administered to a patient.

REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.62/314,316, filed Mar. 28, 2016, which is hereby incorporated byreference in its entirety.

BACKGROUND

Aspects of the present disclosure relate generally to viable cellularcompositions and methods of their preparation, storage and use.

Administration of cellular compositions to humans and animals in thetreatment of various pathologies or disorders has become increasinglyprevalent and bears hope to improve a multitude of therapies. Theability to stably store viable cells and to then prepare them anddeliver them to patients is important. Additives to the compositions forcryopreservation must be effective to preserve viability and alsobiologically acceptable when ultimately delivered to the patient. Aswell, to facilitate their broad distribution and use, it is highlybeneficial if the compositions can be effectively stored through a rangeof conditions that are achievable without undue capital or other expensein the manufacturing and/or distribution chain.

In view of the background in the area, there remain needs for improvedand/or alternative methods and compositions related to storage-stablecell preparation, and their distribution, storage and ultimate use.Aspects of the present disclosure are addressed to these needs.

SUMMARY

It has been discovered that highly robust, storable, viable cellpreparations can be achieved when incorporating controlledconcentrations of additives that help to preserve viability duringcryopreservation, but which ultimately provide easy access totherapeutic, viable cell compositions for administration to patients.Accordingly, in one aspect, the present disclosure provides a method formaking, storing and preparing viable cells that can be administered to apatient. The method includes preparing an unfrozen liquid compositionincluding about 500,000 viable cells/ml to about 20 million viablecells/ml and an aqueous cryopreservation medium includingdimethylsulfoxide at a concentration of about 1 to 3% and hydroxyethylstarch at a concentration of about 2% to 10%. The unfrozen liquidcomposition is sealed in a cryopreservation container. Thecryopreservation container is stored in a freezer at a temperature inthe range of about −60° C. to −100° C. The cryopreservation container isremoved from the freezer and thawed to provide a thawed viable cellcomposition having viable cells suspended in the aqueouscryopreservation medium. A diluting aqueous liquid medium is combinedwith the thawed viable cell composition, for example in a volume ratioof at least 5:1, to provide a diluted viable cell composition containinghydroxyethyl starch and dimethylsulfoxide, with the dimethylsulfoxidepresent in the diluted viable cell composition at a concentration ofabout 0.5% or less. The diluted viable cell composition can also containthe hydroxyethyl starch at a concentration that can be greater than theconcentration of the dimethylsulfoxide and/or that can be at least 0.1%.The diluted viable cell composition can be administered to a patient.

In another aspect, provided is a method for treating a patient withviable cells. The method includes thawing a cryopreserved compositionthat includes viable cells and an aqueous cryopreservation medium,wherein the cryopreservation medium of the cryopreserved compositioncontains dimethylsulfoxide at a concentration of about 1% to 3% andhydroxyethyl starch at a concentration of about 2% to 10%, to provide athawed viable cell composition having the viable cells suspended in anaqueous liquid. The method also includes combining a diluting aqueousliquid medium with the thawed viable cell composition to provide adiluted viable cell composition containing hydroxyethyl starch anddimethylsulfoxide, wherein the dimethylsulfoxide is present in thediluted viable cell composition at a concentration of about 0.5% orless. The method also includes administering the diluted viable cellcomposition to a patient.

In another aspect, provided is a method for preparing viable cells thatcan be used to treat a patient. The method includes thawing acryopreserved composition that includes viable cells and an aqueouscryopreservation medium, wherein the cryopreservation medium of thecryopreserved composition contains dimethylsulfoxide at a concentrationof about 1% to 3% and hydroxyethyl starch at a concentration of about 2%to 10%, to provide a thawed viable cell composition having the viablecells suspended in an aqueous liquid. The method also includes combininga diluting aqueous liquid medium with the thawed viable cell compositionto provide a diluted viable cell composition containing hydroxyethylstarch and dimethylsulfoxide, wherein the dimethylsulfoxide is presentin the diluted viable cell composition at a concentration of about 0.5%or less.

In another aspect, provided is a method for treating a patient withviable cells. The method includes storing a cryopreservation containerin a freezer at a temperature in the range of about −60° C. to −100° C.,the cryopreservation container containing a cryopreserved compositionthat includes viable cells and an aqueous cryopreservation medium,wherein the cryopreservation medium of the cryopreserved compositioncontains dimethylsulfoxide at a concentration of about 1% to 3% andhydroxyethyl starch at a concentration of about 2% to 10%. Thecryopreserved composition is thawed to provide a thawed viable cellcomposition having the viable cells suspended in an aqueous liquid, theaqueous liquid having the dimethylsulfoxide at a concentration of about1% to 3% and the hydroxyethyl starch at a concentration of about 2% to10%. A diluting aqueous liquid medium is combined with the thawed viablecell composition to provide a diluted viable cell composition containinghydroxyethyl starch and dimethylsulfoxide, wherein the dimethylsulfoxideis present in the diluted viable cell composition at a concentration ofabout 0.5% or less. The diluted viable cell composition is administeredto a patient.

In a further aspect, provided is a method for treating a patient withviable cells. The method includes administering a viable cellcomposition to the patient, the viable cell composition containingviable cells suspended in an aqueous liquid containing dimethylsulfoxideat a concentration of 0.5% or less and hydroxyethyl starch at aconcentration that can be greater than the concentration ofdimethylsulfoxide and/or can be at least 0.1%.

In another aspect, provided is a viable cell product including acryopreservation container containing a cryopreserved composition thatincludes viable cells and an aqueous cryopreservation medium, whereinthe cryopreservation medium of the cryopreserved composition containsdimethylsulfoxide at a concentration of about 1% to 3% and hydroxyethylstarch at a concentration of about 2% to 10%.

In another aspect, provided is a method for maintaining viable cells.The method includes maintaining in a cryopreserved condition acomposition that includes viable cells and an aqueous cryopreservationmedium, wherein the cryopreservation medium contains dimethylsulfoxideat a concentration of about 1% to 3% and hydroxyethyl starch at aconcentration of about 2% to 10%.

In another aspect, provided is a viable cell composition foradministration to a patient. The viable cell composition includes anaqueous medium comprising dimethylsulfoxide at a concentration of lessthan 0.5%, and hydroxyethyl starch at a concentration that can begreater than that of the dimethylsulfoxide and/or can be at least 0.1%.The cellular composition also includes viable cells suspended in theaqueous medium, preferably at a concentration of about 200,000 cells/mlto about 10 million cells/ml.

In another aspect, provided is a viable cell composition. Thecomposition includes viable cells and an aqueous cryopreservationmedium. The aqueous cryopreservation medium contains dimethylsulfoxideat a concentration of about 1% to 3% and hydroxyethyl starch at aconcentration of about 2% to 10%. In various embodiments, thecomposition can be in liquid form or in a cryopreserved condition, forexample at a temperature of about −60° C. or lower, for example in somevariants at a temperature of about −60° C. to −100° C.

Additional embodiments of the present disclosure, as well as featuresand advantages thereof, will be apparent from the descriptions herein.

DETAILED DESCRIPTION

Reference to certain embodiments will be made in this detaileddescription and specific language will be used to describe theembodiments. It will be understood that this description is intended tobe illustrative. Any alterations and further modifications in thedescribed embodiments, and any further applications of the principlesthereof, are contemplated as would normally occur to one skilled in theart to which this disclosure pertains.

As disclosed above, in certain aspects the present disclosure relates toviable cell compositions and to related methods of preparation, storageand use. In the discussions that follow, a number of potential featuresor combinations of features are disclosed. It is to be understood thateach such disclosed feature or combination of features can be combinedwith the generalized features discussed in the Summary above, or in theList of Certain Disclosed Embodiments below, to form a disclosedembodiment of the present invention. In addition, all percentages givenherein are given as percentage by weight, unless otherwise stated.

A wide variety of cell types may be used in embodiments of the presentdisclosure. For example, the cells can be skin cells, skeletal musclecells, cardiac muscle cells, lung cells, mesentery cells, adipose cells,or stem cells such as mesenchymal stem cells. Adipose cells may be fromomental fat, properitoneal fat, perirenal fat, pericardial fat,subcutaneous fat, breast fat, or epididymal fat. In certain embodiments,the cells comprise stromal cells, stem cells, or combinations thereof.As used herein, the term “stem cells” is used in a broad sense andincludes traditional stem cells, adipose derived stem cells, progenitorcells, preprogenitor cells, reserve cells, and the like. Exemplary stemcells include embryonic stem cells, adult stem cells, pluripotent stemcells, neural stem cells, liver stem cells, muscle stem cells, muscleprecursor stem cells, endothelial progenitor cells, bone marrow stemcells, chondrogenic stem cells, lymphoid stem cells, mesenchymal stemcells, hematopoietic stem cells, central nervous system stem cells,peripheral nervous system stem cells, and the like. Additionalillustrative cells which can be used include hepatocytes, epithelialcells, Kupffer cells, fibroblasts, neurons, cardiomyocytes, myocytes,chondrocytes, pancreatic acinar cells, islets of Langerhans, osteocytes,myoblasts, satellite cells, endothelial cells, adipocytes,preadipocytes, biliary epithelial cells, and progentior cells of any ofthese cell types.

When used, mesenchymal stem cells (MSC) can be obtained from anysuitable tissue. These include as examples MSCs derived from dentaltissue (such as those harvested from dental pulp, periodontal ligaments,or other dental tissues), testicle tissue, bone marrow; peripheralblood, placental tissue, uterine tissue (including endometrialregenerative cells), umbilical cord blood, umbilical cord tissue, orskin tissue (including full thickness skin tissue). These or other MSCscan be used in aspects of the present disclosure. The MSCs can begenerally an adherent cell population expressing markers CD90 and CD105(>90%) and lacking expression of CD34 and CD45 and MHC class II (<5%) asdetected by flow cytometry.

The cells used in the embodiments herein can be from any suitablespecies of animal, for example a mammal, such as a human, canine (e.g.dog), feline (e.g. cat), equine (e.g. horse), porcine, ovine, caprine,or bovine mammal.

The cells, in a viable state, will be combined with an aqueouscryopreservation medium that includes dimethylsulfoxide (DMSO) and acarbohydrate which in preferred embodiments is hydroxyethyl starch(HES). HES and DMSO are readily available commercially. The combinationof the cells with the DMSO and HES can be conducted in any suitablefashion, including by combining the viable cells with a solutioncontaining both DMSO and HES, or by combining the cells with separatesolutions, one of which contains the DMSO and another of which containsthe HES. These and other modes of providing the viable cells in theaqueous cryopreservation medium will be suitable. As well, it will beunderstood that the cryopreservation medium can contain agents otherthan the DMSO and HES, including other cryopreservation agents. However,in preferred forms, the DMSO and HES will constitute at least 50%, atleast 70%, at least 80%, at least 90%, essentially all (98% or more) orall of the cryopreservation medium other than the water.

In preferred prepared viable cell compositions to be cryopreserved, thecryopreservation medium will include the DMSO at a concentration ofabout 5% or less or about 3% or less, and most preferably in the rangeof about 1% to 3% or in the range of about 1.5% to about 2.5%; and, willinclude the HES at a concentration of about 12% or less or about 10% orless, and most preferably in the range of about 2% to 10% or in therange of about 4% to about 8%. In certain preferred forms, thecryopreservation medium includes DMSO at a concentration of about 2% andHES at a concentration of about 6%.

In preferred prepared viable cell compositions to be cryopreserved, theviable cells will be present at a concentration of at least about200,000 cells/ml, at least about 500,000 cells/ml, at least about 1million cells/ml, at least about 5 million cells/ml, or at least about10 million cells/ml; and/or the viable cells will be present at aconcentration not exceeding about 50 million cells/ml, not exceedingabout 30 million cells/ml, or not exceeding about 20 million cells/ml.In certain variants, the viable cells will be present in the cellcomposition at a concentration in the range of about 500,000 to about 20million cells/ml, or in the range of about 3 million to about 20 millioncells/ml, or in the range of about 5 million to about 15 millioncells/ml, or in the range of about 8 million to about 12 millioncells/ml. In some forms, the viable cells will be present in the viablecell composition at a concentration of about 10 million cells/ml.

The viable cell composition including the viable cells and theabove-disclosed cryopreservation medium, in unfrozen (liquid) form, canbe prepared in, delivered into, or otherwise provided in acryopreservation container such as a cryopreservation vial orcryopreservation bag. In preferred forms, the cell composition isprovided in a cryopreservation vial having features as disclosed in U.S.Pat. No. 8,709,797 issued Apr. 29, 2014, which is hereby incorporated byreference. Preferred cryopreservation vials will thus include an accessport, preferably a needle septum, through which the viable cells can besterilely accessed with a needle or other cannulated device after aperiod of storage (e.g. in cryopreserved state) in the vial. Thepreferred vials will also include two additional ports to the interiorof the vial, which can be used as filling and vent ports (the vent portpreferably containing a sterile filter). These two additional ports canbe fitted with heat sealable tubing, which can be heat sealed to sealthe vial after filling, and which can thereafter be cut prior toaccessing the cells in the vial, for example to provide a vent forneedle-based removal of the cell composition through the needle septum.In preferred forms, each cryopreservation container will contain about 1ml to about 10 ml of the cell composition, more preferably about 1 ml toabout 5 ml of the composition, and most preferably about 2 ml to about 3ml of the composition. Preferred cryopreservation vials for use inembodiments herein are commercially available as CellSeal® CryogenicVials (Cook Regentec LLC, Bloomington, Ind.).

The cryopreservation container containing the viable cell compositioncan be subjected to cryopreservation conditions to cause the compositionto be cryopreserved. For example, the cryopreservation conditions caninclude storing the cryopreservation container at a temperature at whichthe viable cell composition freezes. For example, the cell compositioncan be maintained in a cryopreserved state at a temperature in the rangeof about −60° C. or lower, for example in the range of about −60° C. to−150° C., more preferably about −60° C. to −100° C., and even morepreferably about −70° C. to −90° C. In certain forms, the cellcomposition can be maintained in a cryopreserved state at a temperatureof about −80° C.±5° C. In most preferred forms, the cryopreservationcontainer is stored in a mechanical freezer (a freezer, typicallyelectrically-powered, that uses a re-circulating refrigerant within anair cooler that exchanges heat from air circulating within the freezerto reduce the temperature of contents within the freezer) to maintainthe cell composition in a cryopreserved state at the statedtemperatures. Advantageously, this avoids the need to use liquidnitrogen tanks for cryopreservation storage, at least during a storagephase when the mechanical freezer is used (e.g. a mechanical freezer inoperation at a point of care location to which the viable cellcomposition is shipped, stored in the mechanical freezer, thawed andthen used, e.g. to treat a patient). Thus, in some embodiments, thecryopreservation container containing the viable cell composition can bestored under cryopreservation conditions (e.g. immersed in liquidnitrogen and/or in a mechanical freezer as disclosed herein) at themanufacturing facility, a distribution facility, or another facilitythat is a distance from a point of care facility, the cryopreservationcontainer containing the viable cells can be shipped to the point ofcare while maintaining cryopreservation conditions (e.g. in a shippingpackage containing a liquid nitrogen system and/or dry ice—frozen carbondioxide), and the cryopreservation container containing the viable cellscan be stored at the point of care (e.g. a veterinary clinic orhospital) under cryopreservation conditions until needed foradministration, preferably within a mechanical freezer as disclosedherein.

Preferred viable cell compositions containing the cells, HES and DMSOexhibit good capacity to maintain the viability of the cells duringstorage at a temperature in the range of about −60° C. to −100° C. Forexample, preferred compositions will lose no more than 20% of theirinitial viable cells, no more than 10% of their viable cells, or no morethan 5% of their viable cells, when stored at a temperature in the rangeof −60° C. to −100° C. for a period of six months, and more preferablywhen stored at a temperature in the range of −60° C. to −100° C. for aperiod of one year. Such storage stabilities under the relativelymoderate cooling conditions enable advantageous product distribution anduse methods in which multiple point of care locations (e.g. 10 or more,or 20 or more locations) can maintain mechanical freezers in which aplurality of cryopreservation containers each containing a cellcomposition as described herein are stored. The stability of theviability of the cells under these storage conditions can provide anacceptable shelf life at the point of care. Immediately prior toadministration (e.g. within 6 hours, or within 2 hours, or within 1hour), a cryopreservation container can be removed from the freezer andthe composition therein thawed for additional manipulation (e.g.dilution as described below) and/or administration.

In some methods herein, to thaw the cell composition, thecryopreservation container can be caused to warm to a temperature atwhich the frozen cell composition reverts to a liquid form. This can beachieved in any suitable manner. For example, the container, remainingsterilely sealed, can be exposed to a gaseous environment (e.g. the roomair environment at the point of care) at room temperature (e.g. about20° C. to 25° C.) to thaw the cell composition. In another form, thecontainer can be incubated in a heated liquid bath to thaw the cellcomposition, for example heated to a temperature in the range of about33° C. to 37° C., preferably about 37° C. The cryopreservation andsubsequent thawing of the cell composition will preferably result inhigh maintenance of cell viability, for example with the thawedcomposition retaining at least 80%, at least 90%, or at least 95% of theviable cells of the initial cell composition prior to cryopreservation.

After thawing, the viable cell composition can be removed from thecryopreservation container. For example, in some embodiments, thecryopreservation container has a septum through which a needle or othercannulated device may be passed to access the container contents, whilemaintaining a sterile environment within the container. The needle orother cannulated device can then be used to withdraw the cellcomposition from the container sterilely, for example into a syringebarrel or other vessel. It will be understood that other methods foraccessing the cell composition within and removing the viable cellcomposition from the cryopreservation container may be used in otherembodiments.

In some forms, the viable cell composition can be administered to apatient, or put to a research or other use, unmodified from itscomposition upon removal from the cryopreservation container. In someembodiments, the viable cell composition is modified for use, includingfor use in administration to a patient. The modification can include,for example, the addition of one or more substances to the viable cellcomposition.

In preferred methods, the viable cell composition is combined with anaqueous liquid diluting medium to dilute the cell concentration of thecomposition, and potentially also dilute the concentration of othercomponents, such as HES and/or DMSO, of the viable cell composition. Inbeneficial aspects, the diluting aqueous liquid medium can be aphysiologically acceptable aqueous liquid, such as a phosphate-bufferedor otherwise buffered saline solution, potentially with other additives.Also, it will be understood that the diluting aqueous liquid medium canbe combined with the viable cell composition as a single volume of agiven composition, or as multiple volumes of the same composition or ofdiffering compositions.

In preferred methods, to prepare a diluted viable cell composition foradministration to a patient or other use, a diluting aqueous liquidmedium will be combined with the cell composition in a volume ratio ofat least 2:1, at least 3:1, at least 5:1, or at least 8:1. In morepreferred forms, such volume ratio will be in the range of about 3:1 toabout 20:1, or about 5:1 to about 15:1. It is contemplated that the cellconcentration, DMSO concentration and HES concentration in the prepareddiluted composition will be correspondingly reduced relative to thestarting cell composition prior to dilution; however, in some forms thediluting aqueous liquid medium may itself contain some concentration ofcells, HES and/or DMSO, albeit typically lower than the concentration inthe starting cell composition so as to result in some dilution of cells,HES and DMSO in the diluted cell composition.

The combination of the thawed cell composition with the diluting aqueousliquid medium can be conducted in any suitable container or vessel. Inbeneficial modes, the combination is conducted in a second container(other than the cryopreservation or other container in which the cellswere stored or held). This second container can include an input port orother input member, for example a septum, for sterile transfer ofmaterials such as the cell composition and/or the diluting aqueousliquid medium into the second container. In some forms, combining thecell composition and diluting aqueous liquid medium can includedelivering both of these into the second container, for example ineither order or simultaneously. In other forms, the second container canbe provided as a pre-manufactured container already containing thediluting aqueous liquid medium in sterile condition, and the cellcomposition can be added to the pre-manufactured container. In any ofthese embodiments, the second container can be a bag and/or can have anoutlet port spaced from the input port or other member for delivery ofthe prepared diluted cell composition from the bag or other container,e.g. for delivery into a patient. The second container can be a baghaving a septum for sterile input of materials and a valved port foroutlet of materials, e.g. as occurs in common saline bags for patienttreatment in medical care. The thawed cell composition, and potentiallythe trehalose-containing medium (if not pre-manufactured in the bag) canbe sterilely delivered into the bag by needle through the septum, andthe prepared diluted cell composition can be sterilely delivered to apatient through the valved port.

In certain embodiments, the diluting aqueous liquid medium will becombined with the viable cell composition, and the prepared diluted cellcomposition will have a DMSO concentration that has been reduced fromits initial level (e.g. any of those levels indicated above for thecryopreserved cell composition) to a diluted concentration of about 0.5%or less, more preferably about 0.3% or less, and in some embodiments toa diluted concentration in the range of about 0.05% to about 0.5% or inthe range of about 0.1% to about 0.3%. In addition or alternatively, thediluting aqueous liquid medium will be combined with the cellcomposition, and the prepared diluted cell composition will have an HESconcentration that is greater than that of the DMSO and/or which is atleast about 0.1%, more preferably at least about 0.3%, and in someembodiments a concentration in the range of about 0.1% to about 6% or inthe range of about 0.3% to about 2%. HES (at 6% concentration) is aknown volume extender for administration to the bloodstream, and thus itis contemplated that the diluting aqueous liquid medium can in someembodiments contain significant levels of HES while nonethelesspreparing a diluted cell composition well suited for administration tothe bloodstream of a human or other animal patient. Thus in some modesof operation, the diluted composition can have a DMSO concentration thatis reduced to a greater extent than the reduction, if any, of the HESconcentration, as compared to the starting cell composition prior todilution. In regard to the cell concentration in the diluted cellcomposition, the diluting aqueous liquid medium will typically notcontain any cells, and the cell concentration of the diluted viable cellcomposition will the lower than that of the starting cell composition,for example in the range of about 100,000 cells/ml to about 20 millioncells/ml, or about 250,000 cells/ml to about 5 million cells/ml, orabout 500,000 cells/ml to about 2 million cells/ml. In certain forms,the cell concentration of the prepared diluted cell composition will beabout 1 million cells/ml.

In certain embodiments herein, the diluting aqueous liquid medium willcontain trehalose. Trehalose, also known as mycose or tremalose, is analpha-linked disaccharide formed by an α,α-1,1-glucoside bond betweentwo α-glucose units. It has a chemical name of(2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxane-3,4,5-triol (IUPACnaming convention). The diluting aqueous liquid medium can contain anysuitable concentration of trehalose for these purposes. In certainaspects, the diluting aqueous liquid medium will contain trehalose at aconcentration of about 1% to about 20% by weight, or about 1% to about10% by weight, or about 2% to about 7% by weight, or about 2.5% to about5% by weight, or about 3% to about 4% by weight. The diluting aqueousliquid medium, whether it contains trehalose as specified herein or not,can also include other components and/or have a specified osmolarity.For example, it can include sodium chloride at a physiologicallyacceptable level, for example at a level in the range of about 0.5% toabout 1.5%, e.g. about 0.9% (isotonic). The diluting aqueous liquidmedium can also include a buffer, for example phosphate buffer, and canhave a pH in the range of about 6 to 8, or about 6.8 to 7.8, or about 7to 7.5. The diluting aqueous liquid medium can also have an osmolarityin the range of 200 to 600 milliosmols per kilogram (mosm/kg), or 250 to500 mosm/kg, or 250 to 400 mosm/kg.

A diluting aqueous liquid medium containing trehalose can be combinedwith the cell composition containing DMSO and HES, to prepare atrehalose-containing diluted cell composition having a suitableconcentration of trehalose, along with HES and DMSO (e.g. with the HESand DMSO in the concentrations for the diluted cell compositionspecified above). This concentration of trehalose in the prepareddiluted viable cell composition, in certain embodiments, is in the rangeof about 1% to about 10% by weight trehalose or about 2% to about 7% byweight, or about 2.5% to about 5% by weight, or about 3% to about 4% byweight. Additionally or alternatively, the concentration of trehalosecan be effective to inhibit clumping of the cells as compared to acorresponding cell composition without the trehalose. Inhibition ofclumping can be observed by the formation of fewer and/or smaller clumpsof cells in the prepared cell composition, for example at a time pointat least ten minutes after preparation of the cell composition, at leasttwenty minutes after preparation of the cell composition, or at leastafter 60 minutes after preparation of the cell composition. The capacityof the trehalose to inhibit clumping for significant periods of timefollowing preparation of the cell composition can, for example, providesufficient time to administer the prepared cell composition to apatient, for example by injecting or infusing the cell composition intothe bloodstream of a patient by venous or arterial access and/or bylocal implantation of the cell composition. In therapeutic applicationsof cell compositions, the composition can be administered to the patientover a relatively prolonged period of time, for example at least 10minutes, at least 20 minutes, or at least 60 minutes.

As disclosed herein, the prepared diluted viable cell composition canhave specified concentrations of cells, DMSO, HES and/or trehalose.Where minimum or maximum concentrations, or ranges of concentrations,are stated, it will be understood that the conditions of the dilution ofthe starting viable cell composition can be controlled to achieve thestated amounts in the prepared diluted viable cell composition. Theseconditions include, for example, the concentrations of components in thestarting viable cell composition prior to dilution, the volume ratio ofthe diluting aqueous liquid medium used relative to the starting viablecell composition, and the concentrations (if any) of the identifiedcomponents in the diluting aqueous liquid medium.

The prepared diluted cell composition can be put to any suitable use,including for example research or therapeutic uses. For therapeutic use,the cell composition may as examples be administered to a human oranimal patient to treat or prevent a disease or condition such asdegenerative bone disease, osteoarthritis, rheumatoid arthritis,polyarthritis, systemic lupus erythematosus, inflammatory bowel disease,atopy, hepatitis, chronic steroid responsive meningitis-arteritis,beagle pain syndrome, degenerative myelopathy, chronic renal failuredisease, dilated and mitral cardiomyopathy, keratoconjunctivitis sicca,immune mediated non-erosive arthritis, immune mediated hemolytic anemia,immune mediated thrombocytopenia, Evans syndrome, intervertebral discdisease, muscle fibrosis secondary to disease or trauma, refractoryconical ulcer, diabetes mellitus, spinal trauma, eosinophilic granulomacomplex, hypertrophic cardiomyopathy, cholangitis, spinal injury,exercise induced pulmonary hemorrhage, rhabdomyolysis, conical ulcer,eczema, multiple sclerosis, muscular dystrophy, spinal injury, diabetesmellitus, hepatitis, myocardial infarction, congestive heart failure, ormuscle fibrosis.

The prepared diluted cell composition can be administered to a patientin any suitable manner In certain forms, the cell composition isdelivered systemically into the bloodstream of a patient, for example bydelivery into a vein or artery. In other forms, the cell composition isdelivered topically to the patient (e.g. in the treatment of atopy orother skin disorders). In still other forms, the cell composition isdelivered to a local implant site in a patient. Any of these or anycombination of these modes of administration may be used in thetreatment of a patient. In certain combination treatments, a firstamount of a prepared cell composition herein can be deliveredsystemically into the bloodstream of a patient, and a second amount of aprepared cell composition herein (e.g. prepared with or separately fromthe first amount and including the same type(s) or a different type(s)of cells) is implanted locally in or near one or more skeletal joints ina patient to treat an arthritic condition, e.g. any of those arthriticconditions identified herein. Also, in patient treatments herein, asingle administration of a prepared cell composition as described hereincan be made in some embodiments, while in others multiple separateadministrations of prepared cell compositions as described herein may bemade over time (e.g. weekly or monthly administrations). In furtherembodiments, the prepared, diluted cell composition can be filteredprior to administration to the patient, e.g. to remove any clumps ofcells that may be present. In certain forms, the cell composition can bepassed through an in-line filter positioned in tubing through which thecell composition is passed into the bloodstream of the patient, e.g.into a vein or artery of the patient. Such a filter can, in certainvariants, have a particle size cutoff of about 200 micrometers (i.e.exclude from passage particles having a maximum cross-sectionaldimension of greater than about 200 micrometers) or lower, or a particlesize cutoff of about 170 micrometers or lower, or a particle size cutoffof about 100 micrometers or lower, while allowing the passage of singlysuspended cells through the filter.

Additional embodiments herein include products useful in preparingdiluted cell compositions as described herein. In one embodiment,provided is a diluting aqueous liquid medium useful for preparing adiluted cell composition. The diluting aqueous liquid medium can containthose components, and in amounts, as specified herein. As well, thediluting aqueous liquid medium can be provided in sterile form in acontainer that is included in a kit. That container may be a vial, bagor other container. In certain forms, the container has the features ofthe “second container” discussed hereinabove in which the diluted cellcomposition can be prepared, including for example having an inlet portor other member (e.g. needle septum) and a separate outlet port asdiscussed above. Kits disclosed herein may include the containercontaining the diluting aqueous liquid medium along with one or moreadditional components, for example including but not limited to a liquidtransfer device such as a syringe and attached or attachable needle, andpotentially also a container containing the starting cell composition tobe used to prepare the diluted cell composition. The containercontaining the starting cell composition can include the cellcomposition in a cryopreserved state (e.g. shipped frozen with the kit)or in a non-cryopreserved (e.g. thawed where the cells were previouslycryopreserved) state, including embodiments in which the cryopreservedcell composition contains HES and DMSO in any of those amounts thereforspecified above. Kits disclosed herein may also include at least onefilter, for example a filter as described above, through which aprepared diluted cell composition can be passed prior to administrationinto a patient, and/or tubing through which the diluted cell compositioncan be passed during administration to a patient.

The following specific Experimental is provided to facilitate a furtherunderstanding of aspects of the present disclosure. It will beunderstood that this Experimental is illustrative, and not limiting, innature.

Example 1 C-URCs Isolation and Culture

Fully intact uteri are obtained from a local low-cost spay-neuter clinicfrom female canines that have presented for ovariohysterectomy. Once thesamples arrive at the laboratory, the ovaries are removed and discardedthen the uterus separated into approximate one gram, full thicknesssections.

A one gram sample is then minced to ≦1 mm³ fragments using a sterilescalpel. The chopped tissue is placed into an enzymatic bath anddigested for 30 min at 37° C. Once digestion is complete, the enzymesare neutralized with culture media (DMEM-HG with 10% fetal bovine serumand 0.25 mg/mL amphotericin B, 100 IU/mL penicillin-G, and 100 mg/mLstreptomycin), centrifuged at 300×g for 5 min and re-suspended in freshculture media. The contents are then strained through a 200 μm sterilemembrane and plated in a 25 cm² flask. After 14 days of culture, thecells are split as Passage 0 (P0) using TrypZean™ solution and cellcounts and viability are assessed using a standard trypan blue dyeexclusion assay and hematocytometer. The resulting cells are termedcanine uterine regenerative cells (C-URCs).

Example 2 C-URCs Cryopreservation, Thawing, and Dilution

C-URC's prepared as in Example 1, or prepared by expanding a cultureprepared as in Example 1 in a bioreactor, are suspended at aconcentration of 10 million cells/ml in a cryopreservation solutioncontaining 2% DMSO and 6% HES, and this cell composition is sterilelyfilled in 3 ml aliquots into respective cryopreservation vials(available as 5 ml capacity CellSeal® Cryogenic Vials from Cook RegentecLLC, Bloomington, Ind.). The vials are sealed and then frozen and storedat −80° C. in a mechanical freezer for one to twelve months.

After its storage period, a vial is removed from the freezer and thawedby immersing the vial in a 37° C. water bath for several minutes. Afterthawing, the sterile filter-containing vent tube of the vial is cut toopen the vent, and a sterile syringe needle is used to penetrate theneedle septum of the vial and withdraw the cell composition into thesyringe barrel. The needle is then removed from the vial's septum, andused to penetrate the septum input port of a sterile bag containing 27ml of a buffered physiologic saline solution containing 3.67% trehalose.The prepared, diluted cell composition has a cell concentration of 1million cells/ml, and DMSO, HES and trehalose concentrations of 0.2%,0.6% and 3.3%, respectively. The prepared diluted cell composition canbe administered to a patient from a valved output port of the bag, forexample through medical tubing having an in-line filter to removeclumped cells. The administration can be intravenous.

List of Certain Disclosed Embodiments

The following provides an enumerated, non-limiting listing of certainembodiments that are disclosed herein.

Embodiment 1. A method for treating a patient with viable cells,comprising:

thawing a cryopreserved composition that includes viable cells and anaqueous cryopreservation medium, wherein the cryopreservation medium ofthe cryopreserved composition contains dimethylsulfoxide at aconcentration of about 1% to 3% and hydroxyethyl starch at aconcentration of about 2% to 10%, to provide a thawed viable cellcomposition having the viable cells suspended in an aqueous liquid;

combining an aqueous liquid diluting medium with the thawed viable cellcomposition, preferably in a volume ratio of at least 2:1 and morepreferably at least 5:1, to provide a diluted viable cell compositioncontaining hydroxyethyl starch and dimethylsulfoxide, wherein thedimethylsulfoxide is present in the diluted viable cell composition at aconcentration of about 0.5% or less; and

administering the diluted viable cell composition to a patient.

Embodiment 2. A method for preparing viable cells for treating apatient, comprising:

thawing a cryopreserved composition that includes viable cells and anaqueous cryopreservation medium, wherein the cryopreservation medium ofthe cryopreserved composition contains dimethylsulfoxide at aconcentration of about 1% to 3% and hydroxyethyl starch at aconcentration of about 2% to 10%, to provide a thawed viable cellcomposition having the viable cells suspended in an aqueous liquid; and

combining an aqueous liquid diluting medium with the thawed viable cellcomposition, preferably in a volume ratio of at least 2:1 and morepreferably at least 5:1, to provide a diluted viable cell compositioncontaining hydroxyethyl starch and dimethylsulfoxide, wherein thedimethylsulfoxide is present in the diluted viable cell composition at aconcentration of about 0.5% or less. Embodiment 3. The method ofembodiment 1 or 2, also comprising, prior to the thawing:

storing a cryopreservation container in a freezer at a temperature inthe range of about −60° C. to −100° C., the cryopreservation containercontaining the cryopreserved composition.

Embodiment 4. The method of any one of the preceding embodiments,wherein the thawed viable cell composition has a concentration of theviable cells in the range of about 1 million cells/ml to about 20million cells/ml, and wherein the diluted viable cell composition has aconcentration of the viable cells in the range of about 200,000 cells/mlto about 5 million cells/ml.Embodiment 5. The method of any one of the preceding embodiments, alsocomprising administering the diluted viable cell composition to apatient by delivering the diluted viable cell composition into a bloodvessel of the patient.Embodiment 6. The method of any one of the preceding embodiments,wherein the cryopreservation medium of the cryopreserved compositioncontains the dimethylsulfoxide at a concentration of about 1.5% to 2.5%.Embodiment 7. The method of any one of the preceding embodiments,wherein said volume ratio is at least 8:1, and wherein thedimethylsulfoxide is present in the diluted viable cell composition at aconcentration of about 0.3% or less.Embodiment 8. The method of any one of the preceding embodiments,wherein the cryopreservation medium of the cryopreserved compositioncontains the dimethylsulfoxide at a concentration of about 1.75% to2.25%.Embodiment 9. The method of any one of the preceding embodiments,wherein the viable cells comprise viable mesenchymal stem cells.Embodiment 10. The method of embodiment 9, wherein the viable cellscomprise viable canine mesenchymal stem cells.Embodiment 11. The method of embodiment 9, wherein the viable cellscomprise viable feline mesenchymal stem cells.Embodiment 12. The method of embodiment 9, wherein the viable cellscomprise viable equine mesenchymal stem cells.Embodiment 13. The method of embodiment 9, wherein the viable cellscomprise viable human mesenchymal stem cells.Embodiment 14. The method of any preceding embodiment, wherein theviable cells are derived from dental tissue, testicle tissue, bonemarrow; peripheral blood, placental tissue, uterine tissue, umbilicalcord blood, umbilical cord tissue, or skin tissue.Embodiment 15. The method of any preceding embodiment, wherein thediluted viable cell composition contains the hydroxyethyl starch at aconcentration of at least about 0.1%, and more preferably at least about0.3%.Embodiment 16. The method of any preceding embodiment, wherein thediluted viable cell composition contains the DMSO at a concentration inthe range of about 0.05% to about 0.5% and the hydroxyethyl starch at aconcentration in the range of about 0.1% to about 6%.Embodiment 17. The method of any preceding embodiment, wherein theaqueous liquid diluting medium comprises trehalose.Embodiment 18. The method of embodiment 17, wherein the trehalose ispresent in the aqueous liquid diluting medium at a concentration ofabout 1% to about 10%.Embodiment 19. The method of embodiment 17 or 18, wherein the trehaloseis present in the diluted viable cell composition at a concentration ofabout 2% to about 7%.Embodiment 20. The method of any one of the preceding embodiments,wherein the cryopreserved composition contains the dimethylsulfoxide ata concentration of about 2% and the hydroxyethyl starch at aconcentration of about 6%.Embodiment 21. A method for maintaining viable cells, comprising:

maintaining in a cryopreserved condition a composition that includesviable cells and an aqueous cryopreservation medium, wherein thecryopreservation medium contains dimethylsulfoxide at a concentration ofabout 1% to 3% and hydroxyethyl starch at a concentration of about 2% to10%.

Embodiment 22. The method of embodiment 21, wherein said maintainingcomprises maintaining the composition at a temperature in the range ofabout −60° C. to −100° C.Embodiment 23. The method of embodiment 21 or 22, wherein saidmaintaining comprises cooling the composition in a mechanical freezer.Embodiment 24. The method of any one of embodiment 21 to 23, wherein thecomposition has a concentration of the viable cells in the range ofabout 1 million cells/ml to about 20 million cells/ml.Embodiment 25. The method of any one of embodiments 21 to 24, whereinthe cryopreservation medium of the composition contains thedimethylsulfoxide at a concentration of about 1.5% to 2.5%.Embodiment 26. The method of any one of embodiments 21 to 25, whereinthe cryopreservation medium of the composition contains thedimethylsulfoxide at a concentration of about 1.75% to 2.25%.Embodiment 27. The method of any one of embodiments 21 to 26, whereinthe viable cells comprise viable mesenchymal stem cells.Embodiment 28. The method of any one of embodiments 21 to 27, whereinthe viable cells are derived from dental tissue, testicle tissue, bonemarrow; peripheral blood, placental tissue, uterine tissue, umbilicalcord blood, umbilical cord tissue, or skin tissue.Embodiment 29. The method of any one of embodiments 21 to 28, whereinthe composition contains the dimethylsulfoxide at a concentration ofabout 2% and the hydroxyethyl starch at a concentration of about 6%.Embodiment 30. A method for treating a patient with viable cells,comprising:

administering a viable cell composition to the patient, the viable cellcomposition containing viable cells suspended in an aqueous liquidcontaining dimethylsulfoxide at a concentration of 0.5% or less andhydroxyethyl starch at a concentration that is greater than that of thedimethylsulfoxide and that is at least 0.1%.

Embodiment 31. The method of embodiment 30, wherein said administeringcomprises delivering the viable cell composition into a blood vessel ofthe patient.Embodiment 32. The method of embodiment 30 or 31, wherein the viablecells comprise viable mesenchymal stem cells.Embodiment 33. The method of any one of embodiments 30 to 32, whereinthe patient is a feline, canine, equine or human patient.Embodiment 34. The method of any one of embodiments 30 to 33, whereinthe viable cell composition also comprises trehalose.Embodiment 35. The method of embodiment 34, wherein the trehalose ispresent in the viable cell composition at a concentration of about 2% toabout 7%.Embodiment 36. The method of any one of embodiments 30 to 35, alsocomprising, prior to said administering, preparing the viable cellcomposition by combining an aqueous liquid diluting medium with astarting viable cell composition containing dimethylsulfoxide at aconcentration of about 1 to 3% and hydroxyethyl starch at aconcentration of about 2 to 10%.Embodiment 37. The method of embodiment 36, wherein said combining isconducted with the aqueous liquid diluting medium and starting viablecell composition in a volume ratio of at least 5:1.Embodiment 38. The method of embodiment 36 or 37, wherein the aqueousliquid diluting medium comprises trehalose.Embodiment 39. A product, comprising:

a cryopreservation container containing a cryopreserved composition thatincludes viable cells and an aqueous cryopreservation medium, whereinthe cryopreservation medium of the cryopreserved composition containsdimethylsulfoxide at a concentration of about 1% to 3% and hydroxyethylstarch at a concentration of about 2% to 10%.

Embodiment 40. The product of embodiment 39, wherein the cryopreservedcomposition is at a temperature in the range of about −60° C. to −100°C.Embodiment 41. The product of embodiment 39 or 40, also comprising: amechanical freezer in which the cryopreservation container is received.Embodiment 42. The product of embodiment 41, wherein a plurality of saidproducts are received in the mechanical freezer.Embodiment 43. A viable cell composition for administration to apatient, comprising:

an aqueous medium comprising dimethylsulfoxide at a concentration ofless than 0.5%, hydroxyethyl starch at a concentration of at least about0.1%; and

viable cells suspended in the aqueous medium at a concentration of about200,000 cells/ml to about 20 million cells/ml.

Embodiment 44. The viable cell composition of embodiment 43, wherein:

the aqueous medium also comprises trehalose.

Embodiment 45. The viable cell composition of embodiment 43 or 44,wherein the viable cells are suspended in the aqueous medium at aconcentration of about 200,000 cells/ml to about 5 million cells/ml.Embodiment 46. The viable cell composition of any one of embodiments 43to 45, wherein the viable cells comprise viable mesenchymal stem cells.Embodiment 47. The viable cell composition of embodiment 46, whereingreater than 90% of the viable cells express markers CD90 and CD105 andwherein less than 5% of the viable cells express markers CD34, CD45 andMHC class II.

1. (canceled)
 2. A method for preparing viable cells for treating apatient, comprising: thawing a cryopreserved composition that includesviable cells and an aqueous cryopreservation medium, wherein thecryopreservation medium of the cryopreserved composition containsdimethylsulfoxide at a concentration of about 1% to 3% and hydroxyethylstarch at a concentration of about 2% to 10%, to provide a thawed viablecell composition having the viable cells suspended in an aqueous liquid;and combining an aqueous liquid diluting medium with the thawed viablecell composition, to provide a diluted viable cell compositioncontaining hydroxyethyl starch and dimethylsulfoxide, wherein thedimethylsulfoxide is present in the diluted viable cell composition at aconcentration of about 0.5% or less.
 3. The method of claim 2, alsocomprising, prior to the thawing: storing a cryopreservation containerin a freezer at a temperature in the range of about −60° C. to −100° C.,the cryopreservation container containing the cryopreserved composition.4. The method of claim 2, wherein the thawed viable cell composition hasa concentration of the viable cells in the range of about 1 millioncells/ml to about 20 million cells/ml, and wherein the diluted viablecell composition has a concentration of the viable cells in the range ofabout 200,000 cells/ml to about 5 million cells/ml.
 5. The method ofclaim 2, also comprising administering the diluted viable cellcomposition to a patient by delivering the diluted viable cellcomposition into a blood vessel of the patient.
 6. The method of claim2, wherein the cryopreservation medium of the cryopreserved compositioncontains the dimethylsulfoxide at a concentration of about 1.5% to 2.5%.7. The method of claim 2, wherein said volume ratio is at least 8:1, andwherein the dimethylsulfoxide is present in the diluted viable cellcomposition at a concentration of about 0.3% or less.
 8. The method ofclaim 2, wherein the cryopreservation medium of the cryopreservedcomposition contains the dimethylsulfoxide at a concentration of about1.75% to 2.25%.
 9. The method of claim 2, wherein the viable cellscomprise viable mesenchymal stem cells. 10-14. (canceled)
 15. The methodof claim 2, wherein the diluted viable cell composition contains thehydroxyethyl starch at a concentration of at least about 0.1%.
 16. Themethod of claim 2, wherein the diluted viable cell composition containsthe DMSO at a concentration in the range of about 0.05% to about 0.5%and the hydroxyethyl starch at a concentration in the range of about0.1% to about 6%.
 17. The method of claim 2, wherein the aqueous liquiddiluting medium comprises trehalose.
 18. The method of claim 17, whereinthe trehalose is present in the aqueous liquid diluting medium at aconcentration of about 1% to about 10%.
 19. The method of claim 18,wherein the trehalose is present in the diluted viable cell compositionat a concentration of about 2% to about 7%.
 20. The method of claim 2,wherein the cryopreserved composition contains the dimethylsulfoxide ata concentration of about 2% and the hydroxyethyl starch at aconcentration of about 6%.
 21. A method for maintaining viable cells,comprising: maintaining in a cryopreserved condition a composition thatincludes viable cells and an aqueous cryopreservation medium, whereinthe cryopreservation medium contains dimethylsulfoxide at aconcentration of about 1% to 3% and hydroxyethyl starch at aconcentration of about 2% to 10%.
 22. The method of claim 28, whereinsaid maintaining comprises maintaining the composition at a temperaturein the range of about −60° C. to −100° C. 23-26. (canceled)
 27. Themethod of claim 21, wherein the viable cells comprise viable mesenchymalstem cells.
 28. (canceled)
 29. The method of claim 21, wherein thecomposition contains the dimethylsulfoxide at a concentration of about2% and the hydroxyethyl starch at a concentration of about 6%.
 30. Amethod for treating a patient with viable cells, comprising:administering a viable cell composition to the patient, the viable cellcomposition containing viable cells suspended in an aqueous liquidcontaining dimethylsulfoxide at a concentration of 0.5% or less andhydroxyethyl starch at a concentration that is greater than that of thedimethylsulfoxide and that is at least 0.1%.
 31. The method of claim 30,wherein said administering comprises delivering the viable cellcomposition into a blood vessel of the patient.
 32. The method of claim30, wherein the viable cells comprise viable mesenchymal stem cells. 33.(canceled)
 34. The method of claim 30, wherein the viable cellcomposition also comprises trehalose.
 35. The method of claim 34,wherein the trehalose is present in the viable cell composition at aconcentration of about 2% to about 7%.
 36. The method of claim 30, alsocomprising, prior to said administering, preparing the viable cellcomposition by combining an aqueous liquid diluting medium with astarting viable cell composition containing dimethylsulfoxide at aconcentration of about 1 to 3% and hydroxyethyl starch at aconcentration of about 2 to 10%.
 37. The method of claim 36, whereinsaid combining is conducted with the aqueous liquid diluting medium andstarting viable cell composition in a volume ratio of at least 5:1. 38.The method of claim 36, wherein the aqueous liquid diluting mediumcomprises trehalose.
 39. A product, comprising: a cryopreservationcontainer containing a cryopreserved composition that includes viablecells and an aqueous cryopreservation medium, wherein thecryopreservation medium of the cryopreserved composition containsdimethylsulfoxide at a concentration of about 1% to 3% and hydroxyethylstarch at a concentration of about 2% to 10%. 40-42. (canceled)
 43. Aviable cell composition for administration to a patient, comprising: anaqueous medium comprising dimethylsulfoxide at a concentration of lessthan 0.5%, hydroxyethyl starch at a concentration of at least about0.1%; and viable cells suspended in the aqueous medium at aconcentration of about 200,000 cells/ml to about 20 million cells/ml.44. The viable cell composition of claim 43, wherein: the aqueous mediumalso comprises trehalose. 45-47. (canceled)